methods for global methylation - what is the choice method? (Apr/16/2007 )
I am looking for a good method to compare global methylation percentage. As I can not find a capillary electrophoresis unit available that is coupled to a laser to use that type of detection, and the only other instrument that I can find is a mass spec with an HPLC. Is there another method anybody can come up with that is sensitive and does not require either a mass spec or capillary electrophoresis? Your input would be great.
I use HPLC with UV detection to measure global methylation levels. I believe this is one of the most accurate methods to measure genomic methylation content as you are actually analysing the entire genome, not just one particular region and making an estimation of global levels from that. Why don't you want to use mass spec? If I could change anything about my system it would be to have a mass spec for detection.
Davo: Could you explain your DNA degradation protocol and your HPLC column, elution, and standards?
Treatment of DNA with RNAse A and RNAse T1. Phenol/chloroform purify. Resuspend in dH20. Use 3ug of DNA and ~1.5 units Nuclease P1. Incubate 16 hours at 37 deg. Add 1 unit CIAP for 2 hours.
Following phenol/chloroform extraction, I resuspend in a very small amount of water to keep the sample concentrated. I aim to get the DNA at about 1ug/ul, I suppose I have a lot of DNA from cell lines to play with, using smaller amounts may be a little harder.
This is basically from a paper by Ramsahoye, PubMed ID 12095275.
My column is a Supelcosil LC-18-DB column, with 0.03M ammonium phosphate buffer with 2-4% methanol, no gradient, just isocratic. Run temp is at 35 degrees.
My standards are dNTP's that I have treated with CIAP. I am currently working on making some standards with a methylcytosine aswell which the dNTPs lack.
Does that cover it all?
Thanks. I notice they are running their column at 10C, and omit the 3% methanol in the mobile phase. What made you shift from these?
mass spec is more variable than HPLC between injections, I think HPLC is the way to go,
otherwise it would be an ELISA based method using anti 5-meC
It was the info sheet that came with the column I think that said to use the buffers I use. I think retention times would be much longer without methanol.
I think the temp also came from the same info sheet. In the Ramsahoye paper it shows the difference between running at ambient temp and 10 deg. I don't think there is a hug difference between the two really. I didn't want to run at ambient temp either - if you can control a variable you should! I have also noticed it is a totally different column to what I am using and the order of elution is different to that seen on my column. The catalog number for the column was 58355U from Sigma. I also used some standards from sigma but found that the elution times differed to the times seen from a real DNA sample. The catalog number for that was 47310U.
Nick: With the variation of mass spec vs HPLC, do you mean a mass spec to detect elution when coupled to a HPLC machine?
I was talking more GC/MS for quantitating amounts as the peak areas seem to be more variable.
I suppose if you have HPLC/MS would tell you what the compound you are looking at with the identified peak that would solve our issue of which peak is which!!
As for not wanting to do HPLC and mass spec, it is not that I don't want to do it, I am ready to go, it is more convincing my boss that is the way to go. He keeps saying that there is some other enzymatic way to do it without producing a reference for a protocol. As for an ELISA and blot method, from my experience doing blots, they are not quantitative enough, colorometric and chemolumenescence are not good for actual quantitation. I know that infrared like a Lycor scanner can be quantitative, but I don't think it is the way to go.
I suppose I am looking for this mystery protocol that my boss is talking about .
Hi bunja, the mystery protocol relates to a HpaII/MspI digestion followed by a single-nucleotide extension, first described by Progribny et al. A non-radioactive version of this assay has been described by Bönsch et al 2004. It is relatively easy to do, but it surely is not as reliable as the HPLC or MS techniques. If you want, I can PM you a protocol.